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1 r Human Brain Mapping 35: (2014) r Reversing Pathologically Increased EEG Power by Acoustic Coordinated Reset Neuromodulation Ilya Adamchic, 1 * Timea Toth, 1 Christian Hauptmann, 1 and Peter Alexander Tass 1,2 1 Institute of Neuroscience and Medicine Neuromodulation (INM-7), J ulich Research Center, J ulich, Germany 2 Department of Neuromodulation, University of Cologne, Cologne, Germany r r Abstract: Acoustic Coordinated Reset (CR) neuromodulation is a patterned stimulation with tones adjusted to the patient s dominant tinnitus frequency, which aims at desynchronizing pathological neuronal synchronization. In a recent proof-of-concept study, CR therapy, delivered 4 6 h=day more than 12 weeks, induced a significant clinical improvement along with a significant long-lasting decrease of pathological oscillatory power in the low frequency as well as c band and an increase of the a power in a network of tinnitus-related brain areas. As yet, it remains unclear whether CR shifts the brain activity toward physiological levels or whether it induces clinically beneficial, but nonetheless abnormal electroencephalographic (EEG) patterns, for example excessively decreased d and=or c. Here, we compared the patients spontaneous EEG data at baseline as well as after 12 weeks of CR therapy with the spontaneous EEG of healthy controls by means of Brain Electrical Source Analysis source montage and standardized low-resolution brain electromagnetic tomography techniques. The relationship between changes in EEG power and clinical scores was investigated using a partial least squares approach. In this way, we show that acoustic CR neuromodulation leads to a normalization of the oscillatory power in the tinnitus-related network of brain areas, most prominently in temporal regions. A positive association was found between the changes in tinnitus severity and the normalization of d and c power in the temporal, parietal, and cingulate cortical regions. Our findings demonstrate a widespread CR-induced normalization of EEG power, significantly associated with a reduction of tinnitus severity. Hum Brain Mapp 35: , VC 2013 The Authors Human Brain Mapping published by Wiley Periodicals, Inc. Key words: tinnitus treatment; desynchronization; electroencephalography; non-invasive neuromodulation; phantom perception r r This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Additional Supporting Information may be found in the online version of this article. *Correspondence to: Ilya Adamchic, Institute of Neuroscience and Medicine Neuromodulation, J ulich Research Center, Leo-Brand- Straße, J ulich, Germany. Received for publication 12 November 2012; Revised 24 February 2013; Accepted 8 April DOI: /hbm Published online 1 August 2013 in Wiley Online Library (wileyonlinelibrary.com). INTRODUCTION Subjective tinnitus is a frequent sensation of sound that cannot be attributed to an external sound source. Tinnitus may sufficiently affect everyday life, leading to sleep disorders, work disability, and psychiatric problems [Gopinath et al., 2010; Langguth et al., 2011; Schutte et al., 2009]. It is generally accepted that tinnitus generation has a central basis, typically being initiated by damage to the peripheral hearing system [De Ridder et al., 2011a,2011b; Eggermont and Roberts, 2004; Weisz et al., 2005, 2006]. VC 2013 The Authors Human Brain Mapping published by Wiley Periodicals, Inc.

2 r Adamchic et al. r In most cases, tinnitus is associated with an audiometrically measurable hearing loss that largely coincides with the tinnitus frequency range [Norena et al., 2002]. However, also in patients with a normal audiogram the presence of tinnitus may be accompanied by abnormal inner hair-cell function [Weisz et al., 2006]. Both human and animal data show that deafferentation alters receptive fields [Dietrich et al., 2001; Irvine et al., 2001; Rauschecker, 2005] and leads to the emergence of pathological neural synchrony [Hauptmann and Tass, 2007; Merzenich et al., 1984; Ochi and Eggermont, 1997; Verkindt et al., 1995; Weisz et al., 2005] in brain regions deprived of peripheral input. Indeed, pathologically enhanced neuronal synchronization was observed in the primary auditory cortex of animals following damage to the inner ear [Hauptmann and Tass, 2007; Merzenich et al., 1984; Ochi and Eggermont, 1997] as well as in tinnitus patients [De Ridder et al., 2011b; Llinas et al., 1999; Weisz et al., 2005, 2007b]. In a magnetoencephalography (MEG) study, Weisz et al. [2005] showed a reduction of the a rhythm and a concomitant increase of slow-wave (d) activity, particularly in temporal regions, in a group of individuals with chronic tinnitus compared to tinnitus free controls. In fact, low-frequency oscillations are typical for cortical regions deprived of afferent input [Steriade, 2006]. However, low frequencies are not, in general, pathological as they regularly occur during slow-wave sleep [Benoit et al., 2000; Steriade, 2006]. In contrast, in patients suffering from chronic subjective tinnitus, one observes persistent low-frequency activity, present in the awake state. Further studies revealed that positive symptoms may arise owing to the concerted action of slow- and high-frequency oscillations [De Ridder et al., 2007; Llinas et al., 1999; Weisz et al., 2007b]. Slowwave activity facilitates and sustains c activity that, in turn, may serve as a neural code of auditory phantom perception [De Ridder et al., 2007; Weisz et al., 2007b]. Clinical significance of this pattern of electroencephalogram (EEG) abnormality is confirmed by the fact that the tinnitus-related distress and tinnitus loudness are correlated with this abnormal spontaneous activity pattern [De Ridder et al., 2011b; van der Loo et al., 2009; Weisz et al., 2005]. Apart from auditory cortical brain areas, nonauditory areas involved in attention and emotional regulation were also shown to be involved in the tinnitus generation, in particular, in patients with considerable amount of tinnitus distress [De Ridder et al., 2011a; Vanneste et al., 2010]. Furthermore, an altered functional connectivity between auditory and nonauditory regions seems to be a hallmark of the auditory phantom perception [De Ridder et al., 2011a; Schlee et al., 2009a]. However, these pioneering reports of altered EEG=MEG rhythmicity in tinnitus were related to a comparison between a group of tinnitus patients and a group of tinnitus free controls [Moazami- Goudarzi et al., 2010; Weisz et al., 2005]. One limitation of these studies is that, contrary to the normal hearing control group, tinnitus patients typically have an audiometrically measurable hearing loss [Weisz et al., 2005]. Consequently, it was unclear whether the observed EEG abnormalities were specific to tinnitus, rather than being generated by the sensory deprivation owing to the hearing impairment [Weisz et al., 2005]. This point is particularly relevant as the findings by Weisz et al. (2005) are, to a certain extent, qualitatively similar to EEG findings obtained during slow-wave sleep [Mikhailov, 1990]. Accordingly, further attempts to study the electrophysiological correlate of tinnitus in humans focused on intervention-related changes of brain oscillations within one patient group (for review see, Langguth et al., 2012), for example, by measuring neurophysiological effects of tinnitus maskers [Kahlbrock and Weisz, 2008], auditory cortex stimulation via implanted electrodes [De Ridder et al., 2011b] or neurofeedback training [Dohrmann et al., 2007b]. In an MEG study, Kahlbrock and Weisz (2008) found a significant reduction of d-band activity in temporal areas during residual inhibition following the offset of tinnitus masker application [Kahlbrock and Weisz, 2008]. However, no tinnitus-free controls were used in these interventional studies. It is not clear whether intervention-induced tinnitus relief is necessarily related to a normalization of the EEG pattern. In principle, brain oscillations might be modified in such a way that tinnitus decreases, whereas EEG patterns are changed, but still remain significantly different compared to healthy controls. In a previous study, we analyzed clinical and EEG changes caused by acoustic Coordinated Reset (CR) neuromodulation within a population of patients with chronic subjective tinnitus [Tass et al., 2012a]. As shown computationally, CR neuromodulation specifically counteracts pathological neuronal synchronization by desynchronization [Tass, 2003]. Changes of neuronal dynamics and synaptic connectivity are strongly linked in dependence on the relative timing of the pre- and postsynaptic spikes by the spike timing-dependent plasticity (STDP) [Gerstner et al., 1996; Markram et al., 1997]. Already in simple neuronal networks comprising STDP, strongly synchronized states with strong synaptic connectivity may stably coexist with desynchronized states with weak synaptic connectivity [Tass and Hauptmann, 2009; Tass and Majtanik, 2006]. CR-induced desynchronization decreases the rate of coincidences and, hence, owing to STDP also the average strength of the synaptic connections [Tass and Hauptmann, 2009; Tass and Majtanik, 2006]. Consequently, the stimulated neuronal population is shifted from a synchronized state with strong synaptic connectivity to a desynchronized state with weak connectivity [Tass and Hauptmann, 2009; Tass and Majtanik, 2006]: The network undergoes an antikindling, that is, it unlearns pathological connectivity and pathological synchrony. As shown in a computational study, according to the underlying biophysics, the long-lasting desynchronization and the unlearning of pathological connectivity (antikindling) can robustly be achieved by means of direct electrical CR stimulation or indirect, that is, synaptically mediated, excitatory, and inhibitory CR stimulation [Popovych and Tass, 2012]. r 2100 r

3 r Reversing Pathological Neural Activity in Tinnitus r Electrical CR neuromodulation caused long-lasting desynchronization in rat hippocampal slice rendered epileptic by magnesium withdrawal [Tass et al., 2009] and sustained longlasting therapeutic aftereffects in MPTP monkeys [Tass et a., 2012], the standard model of experimental parkionsonism. To specifically counteract pathological neuronal synchrony in the central auditory system, we used acoustic CR neuromodulation [Tass et al., 2012a]. To this end, based on the tonotopic organization of the central auditory system, sequences of pure tones with pitches centered around the patient s dominant tinnitus frequency are periodically delivered in an ON OFF protocol (METHODS) [Tass and Popovych, 2012; Tass et al., 2012a]. Treatment with CR neuromodulation resulted in a highly significant decrease of tinnitus symptoms as measured by visual analog scale (VAS) and tinnitus questionnaire (TQ) scores [Tass et al., 2012a]. According to VAS [Adamchic et al., 2012a] and TQ [Adamchic et al., 2012b] evaluation studies, this improvement is not only statistically, but also clinically significant. In contrast, placebo treatment did not lead to any significant changes. Furthermore, after 12 weeks of acoustic CR neuromodulation d and c activity were significantly decreased in primary and secondary auditory cortex and in frontal areas combined with an increase of the initially reduced a power in auditory and prefrontal areas [Tass et al., 2012a]. However, in that study, EEG markers were compared in one patient population before and after CR therapy [Tass et al., 2012a]. Strictly speaking, it was, thus, not possible to judge whether the therapy shifted the EEG markers closer to what is supposed to be physiological (as represented by a control group of healthy subjects) or whether a completely different pattern of EEG markers evolved, for example characterized by significantly reduced d and=or c power as opposed to controls. Although neural synchronization plays a fundamental role in the pathophysiology of the auditory phantom perception, up to our knowledge, as yet no study has investigated therapy-induced changes of EEG patterns in tinnitus patients as compared to physiological reference EEG patterns recorded from a group of tinnitus-free controls. In this study, we set out to overcome this shortcoming. To further our understanding of the pathophysiology of chronic subjective tinnitus and of the mechanisms of acoustic CR neuromodulation, the goals of this study are as follows: (i) To statistically discriminate between na ıve tinnitus patients and tinnitus-free controls on the basis of EEG spectral parameters. (ii) To compare the EEG pattern in the tinnitus patient population after 12 weeks of CR therapy with the EEG pattern of the tinnitus-free controls. (iii) To explore relationships between CR therapy-induced changes of different resting EEG parameters (i.e., power changes in different frequency bands observed in different brain areas) on the one hand and tinnitus symptoms on the other hand. In particular, to study whether acoustic CR neuromodulation normalizes the EEG pattern in the tinnitus patients (i.e., shifts the EEG patterns closer to a physiological pattern), or whether clinical improvement is associated with a significantly different, nonphysiological EEG pattern. To our knowledge, this is the first study that investigates whether treatment-induced long-lasting changes of resting EEG spectral parameters contribute to a normalization of oscillatory brain activity and if so in which cortical brain regions normalization of EEG spectral parameters takes place. To test this, we combined a cross-sectional with a longitudinal approach. To assess the relationship between the normalization of oscillatory activity in different brain areas and the reduction of tinnitus severity, we used the partial least-squares (PLS) multivariate approach that holds specific advantages over conventional univariate approaches [McIntosh et al., 1996]. MATERIALS AND METHODS Patients Here, we analyze EEG data recorded in tinnitus patients who participated in a multicentric randomized, controlled clinical trial on Acoustic CR Neuromodulation in the Treatment of chronic subjective tonal tinnitus, performed in Germany between 2009 and 2010 ( RESET study, ClinicalTrials.gov Identifier: NCT ). In total, 63 patients with chronic subjective tonal tinnitus participated in the RESET study. All patients were informed about the scope and aim of the study and a written consent was obtained from all tinnitus patients according to the declaration of Helsinki and the study was approved by the ethics commission. Patients with pulsatile, ringing, buzzing, roaring, or hissing tinnitus as well as patients with a need for hearing aids, a history of auditory hallucinations, Meniere s disease, diagnosed neurological or mental disorders, and patients taking CNS-acting medication or participating in other tinnitus therapy programs were not included in the study [Tass et al., 2012a]. The extent of the hearing loss was investigated with a pure tone audiogram. Patients with a hearing loss at any of the tested frequencies (i.e., 0.125, 0.250, 0.750, 1, 2, 3, 4, 6, 8, 12 and khz) >50 db were not included in the study. By the same token, patients who were not able to hear all CR therapy tones (see below) were excluded from the study. In brief, 1 4 and 6 12 khz pure-tone averages (PTAs) were calculated. From 63 randomized patients, 61 had EEGs recorded at baseline and 12 weeks. In all, 11 out of these 61 patients were excluded from the analysis as their EEG recordings were performed with lower hardware filter settings ( Hz), not allowing for an analysis of higher c-band activity. Unilateral and bilateral tinnitus patients can have different EEG abnormalities [Vanneste et al., 2011a]. Accordingly, to avoid an influence of such differences, we selected only the patients with bilateral tinnitus (n 5 28). An overview of the patient s baseline characteristics is summarized in Table I. r 2101 r

4 r Adamchic et al. r TABLE I. Baseline characteristics of all patients and the patients with a good clinical response (i.e., TQ improvement 12 points) All patients (n 5 28) CR Treatment In the RESET study, patients were stimulated for 12 weeks using a portable acoustic device and comfortable earphones (for a more detailed description of the CR neuromodulaton treatment, see Tass et al., 2012a). Visits took place after 1, 4, 8, 12, and 16 weeks. Data for this article were obtained from the baseline and the 12-week visit, because EEG recordings for all patients were performed off-stimulation (i.e., at least 2.5 h after cessation of CR neuromodulation) at these visits. Subjectively perceived tinnitus loudness and tinnitus annoyance were assessed off-stimulation using a visual analog scale for loudness (VAS-L) and annoyance (VAS-A). In general, the CR treatment resulted in a highly significant and clinically relevant decrease of tinnitus severity as measured by VAS-L=VAS-A and TQ scores [Adamchic et al., 2012a,2012b; Tass et al., 2012a]. Healthy Controls Patients with a good clinical response (n 5 12) Age (years) (SD) 50.0 (10.5) 49.3 (8.9) Tinnitus duration 6.1 (4.7) 7.8 (5.6) (years) (SD) TQ (SD) 45.6 (16.8) 52.3 (17.5) VAS-L (SD) 68.4 (19.2) 72.1 (20.7) VAS-A (SD) 67.7 (20.5) 71.7 (23.1) 1 4 khz pure-tone (9.99) (10.32) average 6 12 khz pure-tone average (17.65) (13.29) The control group consisted of 16 healthy tinnitus-free subjects (10 men and 6 women) age matched (mean age 45.0, SD 12.5; P ) to the group of 28 tinnitus patients selected for the EEG analysis. Recruitment of study participants, included in the control group, was performed by advertisement and all participants signed informed consent form. Participants, selected to the control group, were screened by physicians for neurological and mental disorders as well as for ear disorders. Subjects taking CNS-acting medication were excluded. The PTAs of 1 4 and 6 12 khz were (13.69) and (19.05), respectively. All healthy subjects were not taking any medication known to affect the EEG. This group was selected to confirm tinnitus-related EEG abnormalities reported previously [De Ridder et al., 2011b; Moazami-Goudarzi et al., 2010; Weisz et al., 2005]. Furthermore, the control group served as a reference for the treatment-induced EEG changes. Patient Groups for EEG Analysis We performed three different comparisons: (i) We compared all 28 patients with bilateral tinnitus before CR therapy with the healthy control group. (ii) We compared all 28 patients with bilateral tinnitus after 12 weeks of CR therapy with the healthy control group. (iii) To investigate symptom-related changes in the oscillatory brain activity, we also investigated a subgroup of patients with a good clinical response defined as TQ improvement > 12 points (n 5 12) as they were expected to display the most pronounced EEG changes. A TQ > 12 cut-off was selected based on the Reliable Change Index [Jacobson and Truax, 1991] with the assumption that it separates patients with moderate to good relief of their tinnitus symptoms from patients with small to no relief in symptoms [Tass et al., 2012a; Turner et al., 2010]. The group of all 28 patients with bilateral tinnitus comprised patients from all therapy Groups G1 G4: 10 from G1, 4 from G2, 6 from G3, and 8 from G4. The group of good responders (n 5 12) consisted of six patients from G1, four from G3, and two from G4. Patients EEG Data Acquisition Every patient underwent two recording sessions: first on the first treatment day before start of the treatment; second at the 12-week visit, minimum 2 h after stopping the last stimulation session. Healthy controls In every healthy control subject, one EEG recording was performed. Patients and healthy controls were instructed to retain from caffeinated beverages on the day of the recording. Patients and controls were seated in an upright position in a comfortable chair. EEG recordings were obtained in a dimly lit room in a Faraday cage. EEG data were collected from 128 surface electrodes (128 channel HydroCel Geodesic Sensor Net). All electrodes were referenced to Cz. The EEG signals were amplified with a Net Amps 200 amplifier (Electrical Geodesis, Eugene, OR), digitized at 1 khz and band-pass filtered from 0.1 to 400 Hz. Recordings were performed during awake state with eyes closed and eyes open alternating 2-min long conditions. We selected the eyes closed data (two 2-min long epochs) for further analysis as they were less affected by artifacts. Subjects were video monitored for behavioral signs of drowsiness and the epochs with signs of drowsiness were excluded from further analysis. We were interested in spontaneous activity; therefore, we excluded the transitions between eyes-open and eyes-closed phases by removing the first and the last 5 s of each eyes-closed epochs. Photogrammetry was performed for all subjects using the Geodesis Photogrammetry system (Electrical r 2102 r

5 r Reversing Pathological Neural Activity in Tinnitus r Geodesis, Eugene, OR) and the individual head shape was modeled for each subject and EEG session. Data Analysis The scalp EEG was rereferenced to average reference. Signals were additionally digitally filtered with a Hz digital filter. Each EEG recording was corrected for blink and eye movements in Brain Electrical Source Analysis (BESA) using the surrogate model approach in BESA (MEGIS Software, 5.2 version) [Ille et al., 2002]. Recordings were further analyzed in MATLAB (The Mathworks, Natick, MA) using EEGLAB ( where artifact rejection was performed. First, epochs containing large non-neural artifacts (e.g., large EMG artifacts, movement artifacts) were removed in EEGLAB. Then, independent component analysis decomposition was performed on the sensor level EEG data [Delorme et al., 2007]. Myogenic components, that is, components containing EMG activity in the absence of any identifiable neurogenic activity, were selected. These components were identified based on the following criteria: (i) high broad peaks around either Hz and higher, (ii) a moderately small and clustered distribution on the topographic maps that mimicked the underlying scalp musculature, (iii) periods of high-frequency activation in the time domain, and (iv) equivalent dipole(s) located outside the brain volume and having a residual variance of 15% or less. Dipole locations were modeled using DIPFIT plug-in in EEGLAB. Head shape and electrode locations were modeled for each subject and EEG session separately using the EGI Photogrammetry system and then imported into the DIPFIT plug-in. Myogenic component selection procedure was performed twice with inter-rater reliability assessed using Krippendorff s alpha (a ). The mean length of the recordings after artifact correction was 3 min 36 s 6 24 s. Surface EEG was transformed into brain source activity using the source montage approach in BESA [Scherg et al., 2002]. A source model was generated with regional neural sources placed in the regions of interest (ROIs) using BESA. The source montage consisted of temporal (T), orbitofrontal (OF), dorsolateral prefrontal cortex (DPFC), and parietal (PA) sources in both hemispheres, one source was also located within the anterior cingulate cortex (CA) and one in the posterior cingulated cortex (CP). We have to point out that the source montage approach used here should not be confused with a fine spatial localization of, for example ERP, activity performed in some functional neuroimaging studies for each patient and condition. Location of regional sources (ROIs) was predefined by the authors based on the results of the previous studies and was the same for every patient and recording [Lanting et al., 2009; Schlee et al., 2009a; Vanneste et al., 2011b; Weisz et al., 2005]. Additional probe-sources were placed into the occipital lobe and in the area of the central sulcus in both hemispheres. Sources outside the ROIs acted as a spatial filter and reduced the contribution of these regions to the ROI. The strength of the source montages approach is that one can obtain time courses of brain activities from distinct brain regions [Scherg et al., 2002]. For the PLS statistical analysis, the temporal source was further subdivided into the two sources: (1) an equivalent dipole modeling the primary auditory cortex (ROI: AC1) with Thalairach coordinates [x,y,z; mm] 640, 226, 212 and orientations: 60.3, 20.5, 20.8 left and right [Verkindt et al., 1995]; (2) the secondary auditory cortex (AC2) was modeled with a dipole having a radial orientation [Hegerl et al., 1994]. Normalized band powers from 5 ROIs (AC1, AC2, OF, DPFC, and PA) within the left and right hemispheres were averaged over hemispheres for each ROI and every patient separately, resulting in seven ROIs for 28 patients [Kahlbrock and Weisz, 2008]. This averaging was justified by standardized low-resolution brain electromagnetic tomography (sloreta) statistical maps that showed a similar spatial distribution of statistically significant differences in both hemispheres. Furthermore, in tinnitus patients altered spontaneous activity pattern in the temporal region was found to be bilaterally symmetrical, and the same holds true also for activation during the acoustic stimulation paradigm [Smits et al., 2007; Weisz et al., 2005]. Thus, no unilateral effects were affected and=or masked through averaging over both hemispheres. The fast Fourier transform was performed on the artifact-free source waveforms after windowing the signal with a 4,096 ms wide cosine squared (cos 2 ) window with 50% overlap. This gave us a frequency resolution of Hz. The following frequency bands were defined: Hz (d), Hz (h), 8 12 Hz (a), Hz (b), Hz (low c), and Hz (high c). In all calculations, we excluded the power line artifact (48 52 Hz). Taking into account the comparatively poor test retest reliability for absolute as compared to relative power bands, we performed our analysis based on the relative power features [John et al., 1983]. The individual power spectra were normalized by dividing power at each frequency by the integral of the power across all frequencies from 1 to 90 Hz. This allowed us to compare subjects with large differences in the overall spectral energy and to estimate the relative contribution of each band to the whole spectrum and to compare the relative contribution of different bands. For the multivariate analysis, power spectra derived with BESA source montage analysis were divided into 1-Hz wide bands in the range from 1 to 90 Hz. As shown in Figures 5 and 6, for a better presentation, these 1-Hz wide bands were labeled by abbreviations consisting of the lower edge of the frequency band followed by the ROI. For instance, 25DPFC stands for the frequency band between 25 and 26 Hz in the DPFC cortex. We used sloreta to confirm the results received with BESA source montage [Pascual-Marqui, 2002]. With sloreta, we computed a three-dimensional linear inverse solution to the EEG inverse problem with a three-shell spherical r 2103 r

6 r Adamchic et al. r head model registered to the Talairach human brain atlas digitized at the Brain Imaging Center of the Montreal Neurological Institute [Pascual-Marqui, 2002]. The solution space was constrained to the gray matter voxels that belonged to cortical and hippocampal regions (a total of 6,430 voxels at a 5-mm spatial resolution). The localization of the differences in current density power between the groups was assessed by voxel-by-voxel t-tests of the slor- ETA images. In the resulting statistical three-dimensional maps, a nonparametric approach was used to identify statistically significant differences of cortical voxels [Nichols and Holmes, 2001]. Briefly, if there were no significant differences between groups, any labeling of voxels would result in an equally likely statistical map. Thus, sloreta statistical maps were randomly relabeled and t-values were recalculated. Under the null hypothesis, each of the t-statistics are equally likely, and thus, the resulting P- value is the proportion of the t-statistic values greater than or equal to the t-statistic of the correctly labeled data (for more details, see Nichols and Holmes, 2001). P-values were derived from 5,000 such permutations. The voxels P- values of 0.05 were colored in a MRI template. Statistical Analysis of Spectra For comparisons of power spectra between groups, a nonparametric Wilcoxon rank sum test for each frequency point was used. This statistical test for frequency point comparisons was corrected for the number of tests conducted using the false discovery rate (FDR, Benjamini and Hochberg, 1995). Multivariate Analysis To determine the relationship between power spectra changes (calculated from BESA ROIs) and changes in clinical scores, we applied the PLS analysis [Krishnan et al., 2011]. Power changes in 1-Hz-wide frequency bands were used for multivariate analysis as predictor variables of changes in TQ scores and VAS values. PLS analysis was selected as it overcomes the problems related to multicollinearity and having too many variables as compared to the number of samples. All 28 patients were included into the PLS model. The power spectra were divided into 1-Hz wide bands starting at 1 to 90 Hz, which yielded a total of 85 1-Hz wide bands. The power in each band was normalized to the range of 1 90 Hz total power. Changes from the first session to the second session were calculated for each 1-Hz band and every patient separately. The power values for the multivariate analysis were structured in an X-matrix with one row per subject (i.e., a total of 28 rows) and one column per 1- Hz-wide frequency band for each source (i.e., a total of 595 columns). The VAS and TQ data were structured into the Y-matrix in an analogous way. For this analysis, we have used VAS-L and VAS-A values as well as TQ total, psychological distress (PD), intrusiveness (I), auditory perceptional difficulties (A), and sleep disturbance (Si). We did not include emotional distress (E) and cognitive distress (C) subscales as they are already represented in PD (PD 5 E 1 C) and somatic complaints (So) as it is difficult to rule out the real origin of somatic complaints (i.e., the Y-matrix consisted of 28 rows and 7 columns). Several dependent variables can be modeled at the same time using PLS allowing for a simpler interpretation of the results. However, if the dependent variables are reasonably independent, computing a single PLS model tends to have many components and to be difficult to interpret. In such a case, a separate modeling of clusters of correlated dependent variables (Ys) results in a set of simpler models with fewer dimensions. Hence, in the process of creating a PLS model one should start with a principal component analysis (PCA) of the matrix of dependent variables (i.e., Y-matrix) to characterize patterns or structure in data [Wold et al., 2004]. If dependent variables cluster in groups in the PCA loading plot, separate PLS models should be considered for each cluster [Wold et al., 2004]. Accordingly, first we performed a PCA on the Y-matrix [Jolliffe, 2002]. The purpose of the PCA analysis was to reveal latent structures existing in the data. This allowed us to identify data components that clustered together and may have a systematic relationship. Accordingly, separate models were needed for each PCA cluster. PCA and PLS techniques share a common basis. Both techniques define the underlying structure of the data (i.e., the latent variables) by projecting linear planes into multidimensional data [Kramer, 1998]. PLS has one important feature compared to PCA: PCA determines latent variables only in one matrix (i.e., predictors), whereas PLS takes into account both the independent (X) and the dependent (Y) variables to build a predictive model. In our case, the PLS approach translates into a predictive model for Y (changes in clinical scores) depending on X (changes in power spectra). Put otherwise, the model describes which components of the power spectra covary with changes in symptoms. Each latent variable will explain a part of the variance in the data. Typically, several components are needed to explain most of the variances in the data. Also, it is desirable to have a smaller number of latent variables, for the results to be more manageable and accountable. Taking this into account, the goal of the PLS regression modeling was to explain the greatest part of the variations in the data with the smallest number of components. PLS was used to model the relationship between the 595 normalized 1-Hz wide power bands as predictive variables on the one hand and the groups of clinical test scores, revealed by PCA, as dependent variables on the other hand. The goodness of fit of the model was evaluated in terms of both R 2 (which is analogous to the Pearson product moment correlation coefficient) and the goodness of prediction Q 2 as determined by a crossvalidation procedure [Vandervoet, 1994; Wold, 1978]. Both R 2 and Q 2 were r 2104 r

7 r Reversing Pathological Neural Activity in Tinnitus r calculated for the original data as well as for 20 random permutations of the dependent variable matrix Y, whereas keeping the X matrix fix. As the matrix of dependent variables [Y 1 ] changes to a new, reordered matrix [Y r ], the correlation between these two matrices (corr[y 1,Y r ]) decreases. If there is a nonrandom relationship between predictors and dependent variable, the predictive PLS model, constructed on the reordered data, should have a predictive power (Q 2 ) that decreases as the correlation between Y 1 and Y r decreases. If this is not the case, and the PLS model for the reordered data shows a similar predictive ability (Q 2 ) as for the initial data, there is a good possibility for the model being based on chance. We excluded less informative and redundant X variables (i.e., variables with low contribution for explaining the Y variable) from the modeling by the variable selection procedure [Marini et al., 2005]. For this purpose, the variable importance in the projection (VIP) was used that has been computed according to the formula used in the SIMCA-P ( Predictive variables that have a VIP value of >1 are most relevant for explaining dependent variables. Accordingly, all descriptors with a VIP value less than 1 were excluded from the model. This procedure was iteratively repeated until an optimal model (the one with the highest Q 2 ) was obtained. RESULTS Clinical Data Mean improvement in the TQ and VAS-L=VAS-A scores for all 28 patients with bilateral tinnitus after 12 weeks of treatment was (P < 0.01) and = (P < 0.01=P < 0.01), respectively. The subgroup of good responders (n 5 12) showed a mean reduction of (P < 0.001) TQ points and = (P < 0.01=P < 0.01) points in VAS-L=VAS-A. At 12 weeks, PTAs in the group of all tinnitus patients were (10.25) for 1 4 khz and (17.80) for 6 12 khz. In the group of good responders at 12 weeks, PTAs were (9.25) for 1 4 khz and (15.21) for 6 12 khz. No significant differences were found between baseline and the 12-week visit PTAs in the tinnitus patients. Significant differences (i.e., P < 0.05) in the hearing thresholds (PTA, 6 12 khz) were found between all tinnitus patients (n 5 28; both at baseline and at 12 weeks) and the control group. Altered EEG Rhythmicity Mean power spectra Let us first consider the EEG at baseline, that is, prior to CR therapy. To summarize the data from all EEG scalp sensors and all subjects, we averaged the individual spectra from all tinnitus patients recorded before CR neuromodulation (n 5 28), from the subgroup of good responders (n 5 12) and from Figure 1. Enhanced d and c EEG power in tinnitus patients. A: In the global EEG power spectrum, d and c power were increased and a power was decreased in tinnitus patients recorded before CR neuromodulation (n 5 28, red line) as compared to the healthy controls (n 5 16, green line). In contrast, the global EEG power spectrum of the group of good responders (n 5 12, blue dashed line) did not differ from that of the group of all patients with bilateral tinnitus before CR therapy. B: Areas indicated with red (i.e., above or below the horizontal line) correspond to statistically significant differences between all bilateral tinnitus patients (n 5 28) and the healthy controls. Significant differences were found in d, low h, a, high b, low and high c bands. the healthy controls (n 5 16), respectively (Fig. 1). The average power spectrum in the group of all bilateral tinnitus patients before CR therapy (n 5 28) showed clear differences from the average power spectrum of the control group. In the patients group, the average spectral power was higher in d (1 3.5 Hz), low h (5 Hz), high b (20 Hz), low c ( Hz), and high c (52 90 Hz) bands. In contrast, the spectral power in the high a band ( Hz) was lower in the tinnitus patients group. In the tinnitus population, the a peak frequency (i.e., the frequency at which the a peak was located) prior to CR therapy was shifted toward lower frequencies as compared to the healthy controls; however, this difference was not significant (P ). The subgroup of 12 good responders (Fig. 1A, blue dashed line) showed the same differences from the healthy population in all frequency ranges as the complete group of 28 bilateral patients before therapy (Fig. 1A, red line). BESA source montage analysis Furthermore, we studied which brain regions contributed most to the significant differences between patient r 2105 r

8 r Adamchic et al. r and control group and at which frequencies. Mann Whitney U tests were performed for each regional source at each frequency point (Fig. 2). The maximal Z-value (jzj 5 3.6) was observed in the 8 12 Hz a band in the temporal regions. Z-values in the temporal region were highest compared to the other ROIs in most of the frequency bands. The subgroup of 12 patients with a TQ improvement of 12 showed no significant differences in spectral power to the subgroup of 16 patients with a TQ improvement of <12 in any of the ROIs: temporal (F , P ), PA (F , P ), DPFC (F , P ), OF (F , P ), anterior cingulate (F , P ), and posterior cingulate (F , P ). We also investigated possible effects of tinnitus duration in patients with a tinnitus history of 4 years and >4 years, respectively [Schlee et al., 2009a]. There were no significant differences in spectral power between patients with tinnitus duration of 4 years and patients with tinnitus duration of >4 years in any ROIs: temporal (F , P ), PA (F , P ), DPFC (F , P ), OF (F , P ), anterior cingulate (F , P ), and posterior cingulate (F , P ). Standardized low-resolution brain electromagnetic tomography sloreta images show significant power differences in different frequency bands between tinnitus patients and healthy controls (Fig. 3). As opposed to the healthy controls, the cortical spectral power of all bilateral tinnitus patients before the start of the treatment was significantly enhanced in the d, b, low c, and high c frequency band. In contrast, in the a band, we observed a reduced cortical spectral power in the tinnitus population as compared to the healthy controls. The reduced a power was found in prefrontal dorsolateral and medial, anterior and posterior cingulate, orbitofrontal, parietal and temporal, and other regions (Fig. 2 and Table II). Spatial peaks (i.e., local maxima) of the spectral power prior to the start of the therapy were localized in the temporal cortex in the d band (Brodman area [BA] 42, t ) and in the temporal and DPFC in the low c band (BA 41, t ; BA 46, t ). Spatial troughs (i.e., local minima) of the spectral power were found in the a band in the temporal (BA 22, t ) and in the DPFC (BA 46, t ). No significant differences between tinnitus patients and healthy controls were found in the h frequency range. Treatment-Induced Changes Furthermore, we studied the effect of acoustic CR neuromodulation on the spontaneous oscillatory brain activity. To this end, first, we investigated the group of all patients with bilateral tinnitus after 12 weeks of CR treatment. The power spectrum of this patient group approached the average spectrum of the healthy control group, with the most prominent changes being observed in the temporal regions (Fig. 2). Consequently, we observed a decrease of the Z-values, indicative of significant differences between tinnitus patients and healthy controls, in a wide range of frequencies (d, a, low and high c) after 12 weeks of CR therapy (Fig. 2). Further, we investigated a group of good responders (n 5 12), that is, patients with a pronounced reduction of their tinnitus symptoms: TQ reduction 12 points. The average EEG spectrum of these patients approached the average spectrum of the healthy controls to an even greater extent than that of all 28 bilateral tinnitus patients and, accordingly, resulted in lower Z-values for d, a, low and high c bands and disappearance of significant differences in h and b bands (Fig. 2). No significant differences were found in any of the ROIs in the group of 12 good responders after 12 weeks of therapy (Fig. 2). Statistical sloreta images reveal a reduction of the number of voxels and, consequently, the number of BAs displaying a significantly different power between all bilateral tinnitus patients after therapy and healthy controls (Fig. 3 and Table II). The number of voxels with power values that significantly differed from those of the healthy population was reduced in the group of all 28 tinnitus patients (Table III). To an even greater extent, the number of voxels with significant power differences decreased in the group of good responders after 12 weeks of CR treatment (Table III). Interestingly, spatial peaks (i.e., local maxima) of the spectral power in the group of all bilateral tinnitus patients after 12 weeks of therapy were still localized in the temporal cortex in the d (BA 41, t ) and low c (BA 41, t ) band. In contrast, in the group of 12 good responders the spatial peaks (i.e., local maxima) of the spectral power were located in the frontal lobe for d (BA 44, t ) and low c (BA 46, t ), whereas the temporal lobes lost nearly all of their significantly different voxels after 12 weeks of CR therapy (Fig. 3). Remarkably, no significant differences were found after treatment in the group of good responders in the b and high c frequency band anymore. A list of BAs displaying significant results according to the sloreta comparison between tinnitus patients versus healthy controls is summarized in the Table II. Association of Changes in Spontaneous Brain Activity with Reduction of Tinnitus Symptoms Principal component analysis The loading plot in Figure 4 shows the results of the PCA on the changes of the VAS and TQ scores. The loading plot shows a clustering of the TQ and VAS according to the similarity of their responses to the therapy. The TQ PD and TQ I (intrusiveness) subscores and the TQ total score turn out to cluster together, whereas VAS-L and VAS-A form another and separate cluster. The TQ A (auditory perceptual difficulties) and TQ Si (sleep disturbances) r 2106 r

9 Power spectra for the group of healthy controls (n 5 16) (green line), the group of all bilateral tinnitus patients (n 5 28) before therapy (red line), the group of all bilateral tinnitus patients (n 5 28) after therapy (orange dashed line), and the subgroup of good responders (n 5 12) (blue line) in the temporal (A), PA (B) and DPFC (C), OF (M), anterior cingulated (N), and posterior cingulated (O) ROIs. The power spectra of all 28 bilateral tinnitus patients before therapy were compared to those of the healthy controls (n 5 16) by means of the Mann Whitney U-test in the temporal (D), PA (E) and DPFC (F), OF (P), anterior cingulated (Q), and posterior cingulated (R) ROIs for each frequency point. In the same manner, the power spectra of all 28 bilateral patients Figure 2. after therapy were compared to the spectra of the healthy controls (n 5 16) (G, H, I, S, T, U). The analogous comparison was also performed between the good responders (n 5 12) and the healthy controls (n 5 16) (J, K, L, V, W, X) in the temporal, PA, DPFC, OF, anterior cingulated, and posterior cingulated ROIs, respectively. Areas indicated with red (i.e., above or below the horizontal line) correspond to statistically significant differences. In plots with neither red areas nor horizontal significance lines, no frequency point attained significance threshold. There is a noticeable trend toward a reduced a peak frequency in the tinnitus population. Supporting Information Figure S1 shows topographical plots of EEG power differences in the specific frequency bands.

10 sloreta functional tomographic maps of the significant differences in the power of regional electric brain activity between all 28 bilateral tinnitus patients before therapy and healthy controls ( Before therapy ), between all 28 bilateral tinnitus patients after 12 weeks of therapy and healthy controls ( After therapy all tinnitus patients n 5 28 ) and between 12 good responders after 12 weeks of therapy and healthy controls ( After therapy good responders n 5 12 ) in five EEG frequency bands d (1 3.5 Hz), a (8 12 Hz), b ( Hz), low c ( Hz), and high c (52 90 Hz). In the h band (4 7.5 Hz) no significant differences were Figure 3. found between tinnitus patients and healthy controls, neither before nor after CR therapy. Voxels of significantly decreased power (P ) in tinnitus patients as compared to healthy controls are labeled in blue, whereas voxels with significantly increased power (P ) are labeled in red. After 12 weeks of acoustic CR neuromodulation, there was a pronounced decrease of number of voxels with power that was significantly different from the healthy controls: In the d, b as well as low and high c band initially pathologically enhanced power decreased, whereas in the a band the initially reduced power reincreased.

11 r Reversing Pathological Neural Activity in Tinnitus r TABLE II. Statistically significant results from the comparison of the group of all bilateral tinnitus patients after 12 weeks of therapy (n 5 28) with healthy controls and the group of good responders after 12 weeks of therapy (n 5 12) with healthy controls a d Left d Right 1, 2, 3, 4, 6, 8, 9, 10, 11, 13, 1, 2, 3, 4, 6, 8,9, 10, 11, 13, 17, 18, 19, 20, 21, 22, 23, 24, 27, 28, 18, 19, 20, 21, 22, 23, 24, 25, 29, 30, 32, 34, 35, 36, 37, 38, 39, 27, 28, 29, 30, 32, 33, 34, 35, 40, 41, 42, 43, 44, 45, 46, 47 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 a Left a Right 1, 2, 3,4,6,8, 9,10, 11, 13, 1, 2, 3, 4, 5, 6,8, 9, 10, 11, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 17, 18, 19, 20, 21, 22, 23, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 25, 27, 28, 29, 30, 31, 32, 33, 36, 37, 38, 39, 40, 41, 42, 43, 44, 34, 35, 36, 37, 38, 39, 40, 41, 45, 46, 47 42, 43, 44, 45, 46, 47 b Left b Right 13, 22, 40, 41, 42 22, 40, 42 Low c left Low c right 1, 2, 3,4,6,8, 9,10, 11, 13, 19, 1, 2, 3, 4, 6,8,9,10,11, 13, 18, 20, 21, 22, 27, 28, 32, 34, 35, 19, 20, 21, 22, 23, 25, 27, 28, 36, 37, 38, 39, 40, 41, 42, 43, 29, 30, 32, 34, 35, 36, 37, 38, 44, 45, 46, 47 40, 41, 42, 43, 44, 45, 46, 47 High c left 13, 22, 40, 41, 42 High c right 13, 20, 21, 22, 38, 41, 42 a BAs comprising at least one significant voxel were included in the table as areas showing significant power differences. Areas not listed here did not show significant power differences between the compared groups. BAs printed in black exhibited significant differences in the group of all bilateral tinnitus patients (before and after therapy) as well as in the group of good responders as compared to the healthy controls, respectively. The power in the black BA was, hence, significantly different from the healthy controls and was not normalized during therapy. BA printed in red were significantly different in the bilateral tinnitus population (n 5 28) from the healthy controls only before the start of the therapy. The power in the red BAs was, thus, significantly normalized in all bilateral patients. BAs printed in blue were significantly different in the group of all bilateral tinnitus patients (n 5 28) from the healthy controls both before and after the therapy, but lost their significance in the group of good responders after 12 weeks of therapy (n 5 12). Hence, the power in the blue BAs was normalized after 12 weeks of therapy in the group of good responders only. subscores were both located at different and, in particular, remote sites compared to the two clusters mentioned above. This indicates that changes in TQ PD and TQ I subscores and the TQ total score on the one hand and VAS-L and VAS-A on the other hand as well as TQ A and TQ Si subscores, respectively, responded differently to the therapy and might be associated with different underlying mechanisms or were connected to similar mechanisms but to a different extent. Based on these results, a further PLS regression modeling was performed, TQ PD and TQ I subscores as well as TQ total scores were analyzed separately from TQ A and TQ Si subscores, being further separated from VAS-L and VAS-A. Partial least-squares First, we performed the PLS for the spectral power variables versus three TQ-dependent variables (TQ PD, TQ I subscores, and TQ total scores). This resulted in a PLS model with good performance (R , Q ). A model validation by permutations indicated the presence of nonrandom associations in the model. The strength of the associations between EEG and TQ scores was determined according to a loading plot (Fig. 5). This loading plot shows the clustering of the predictor variables and the dependent variables, indicating the strength of the association (covariance) between changes in neuronal power with changes in the individual TQ scores. The loading plot combines both PLS components included into the model. The widest positive association was found between the changes in TQ scores and the changes in AC1 b, low and high c activity. Numerous frequency bands in other sources were also positively associated with the changes in symptoms as assessed by the TQ (sub-)scores (Fig. 5). Other frequency bands (located in the left lower quadrant of Fig. 5) were negatively associated with the TQ PD and TQ I subscores and the TQ total scores. A similar model performance was obtained for the PLS model with respect to changes of the VAS-L and VAS-A scores. Two components with 67 predictors resulted in a model with good performance (R , Q ). Figure 6 shows the loading plot with the VAS-L and VAS- A as dependent variables and the frequency bands as predictor variables. The most pronounced positive associations with the changes in the VAS were found for the changes in the following sources and frequency bands: ACI and ACII high d/low h, b, low and high c. Frequency bands in the left bottom corner of the Figure 6 were negatively associated with the VAS-L and VAS-A. Interestingly, in the PLS model for the VAS scores, as opposed to the PLS model for the TQ scores, we found a higher influence of limbic areas (CA and CP) on the clinical VAS scores (Fig. 6). No satisfactory PLS models were found for TQ A and TQ Si subscores. No significant correlations were found between hearing thresholds (PTAs) and power in any of the frequency bands. TABLE III. Reduction of number of voxels showing a significant power difference compared to the healthy controls All 28 patients (%) 12 good responders (%) d a b Low c High c r 2109 r